Mesenchymal stem cells are pluripotent blast or embryonic-like cells located in blood, bone marrow, dermis and perisosteum. In general these cells are capable of renewing themselves over extended periods of time as well as, under various environmental conditions, differentiating into cartilage, bone and other connective tissue. Recently, various investigators have researched the potential for using these cells to repair or regenerate target tissues, e.g., bone, cartilage, etc. In this manner MSCs have been reported to have regenerative capabilities in a number of animal models. See Acosta et al. (2005) Neurosurg Focus 19(3):E4; Barry (2003) Novartis Found Symp. 249:86-102, 170-4, 239-41; Brisby et al. (2004) Orthop Clin. North Am. 35(1):85-89; Buckwalter and Mankin (1998) Instr Course Lect. 47:487-504; Caplan (1991) J Orthop Res. 9(5):641-650. Further, these finding are being extended in clinical trials to humans, however, most of these trials require in vitro expansion of isolated, non-autologous MSCs using highly concentrated recombinant cytokines and growth factors. For example, most human studies have utilized isolated MSCs from bone marrow (or peripheral blood), followed by ex-vivo expansion of the cells in a laboratory setting using a fetal bovine serum (FBS) based culture medium spiked with various recombinant growth factors. These supplemented FBS-based culture mediums have shown the capacity to support MSC expansion but also include the risk of cross-contamination of infectious vectors, use of non-Food and Drug Administration (FDA) approved drugs/factors, e.g., recombinant TGF-β, FGF, cross species reactions, and possible increased potential for forming cancerous progenitors.
In addition, most of the MSC-based human studies have required trained laboratory staff and laboratory equipment to perform the expansion of the isolated MSCs. These techniques are not amenable to performance by physicians and/or hospital staff, especially given that physicians are legally bound by FDA protocols and procedures concerning non-FDA approved drugs. Therefore, it is difficult for MSC based therapies to be performed in a pragmatic manner, i.e., in a hospital setting with hospital employees. Given these numerous concerns, most MSC based research is directed at non-autologous cells that have been isolated and cultured into permanent cell lines.
Doucet (Doucet, Ernou et al. 2005 J. Cell Physiol 205(2):288-36) has recently described a technique for expanding MSCs of young healthy donors using a 5% platelet lystate enriched culture medium. However, Doucet's investigations did not determine effectiveness of these procedures on elderly patients, patients with degenerative joint diseases (for example osteoarthritis), or other patient specific characteristics. Nor was the study performed using any expansion conditions except for 5% platelet lysate enriched culture medium. In this light, it has been shown that there is a wide variation in MSC growth in patients with and without osteoarthritis, with age, with gender, and based on certain genetic phenotypes. Therefore the Doucet study has very limited applicability to real life situations, where most patients in need of MSC-based therapy are generally either older, or have degenerative joint, organ, or spinal diseases. The Doucet data also does not apply to other disease states of bony metabolism such as avascular necrosis or osteonecrosis. In addition, the generalized findings in Doucet are not gender or age specific, having little guidance on how to treat the different sexes or how to expand MSC's from patients of advanced age.
MSC's can readily differentiate in culture depending on cytokine exposure, environmental conditions (pressure, attachment opportunities, passage treatment, etc. . . . ), or other chemical exposure. For example, exposure to varying levels of TGF-beta, FGF, and/or PDGF can all have impacts on the final cell phenotype produced in culture. In addition, leaving cells in culture longer has impacts on differentiation potential. Cells can be cultured for a certain visual morphology, confluence, or density, all of which impacts the final cell product produced and its potential for certain types of tissue repair. As a result, this invention focuses on controlling factors/parameters so as to produce a homogeneous cell product with certain restorative properties.
In replacing or repairing tissue with MSC's, one concern is the use of non-autologous cells. While MSC's have been traditionally considered immune privileged, recent investigations have demonstrated their activation of the natural killer cell system in a foreign host. (Spaggiari, Capobianco et al. 2006 Blood 107(4): 1484-90) This makes the use of non-autologous cells difficult, as it is anticipated that the host's immune system will attack these foreign cells and potentially decimate the population of transplanted MSCs, thus severely limiting their repair capabilities. In addition, a recent work published by Ueda may have other far reaching implications for the use of non-autologous cells. (Ueda, Inaba et al. 2007 Stem Cells 25(6):1356-63) This study demonstrated that senile mice with osteoporosis transferred that disease into normal mice through a bone marrow vector. This suggests that the MSC's of the senile mice with osteoporosis once transferred to normal healthy mice were able to transfer that disease state into normal healthy mice. This genetic vector for disease transmission is concerning, as any donor MSC's would theoretically need to be screened for all known genetic susceptibilities and diseases that may be transferred by the donor.
There is a need in the art for MSC expansion techniques that do not use drugs or growth factors which are not FDA approved and can be effectively used to replace tissue in a patient in need thereof. This replacement should be with autologous cells that have been optimally expanded based on the patient's medical condition, age, gender and other relevant replacement conditions. In addition, there is a need for autologous techniques to yield a homogeneous cell line with known regeneration capabilities and rigid quality control.
The present invention is directed toward overcoming one or more of the problems discussed above.